Invasive Aspergillosis caused by azole resistant A. fumigatus has a mortality rate nearing an alarming 90%, making this a clinical problem of acute significance. Early work on A. fumigatus suggested that azole resistance was rare and that the genetic basis of resistance was most often due to changes in a gene (cyp51A) encoding the azole target protein, lanosterol ?-14 demethylase. Recent work from our groups has provided evidence that expression of an ATP-binding cassette (ABC) transporter-encoding gene called abcG1 (aka cdr1B) is linked to azole resistance in the absence of any changes at the cyp51A locus. Here we propose to investigate a key transcriptional regulator called AtrR that coordinately regulates expression of both cyp51A and abcG1. AtrR is a Zn2Cys6 zinc cluster-containing factor that resembles other fungal transcriptional regulators of drug resistance. We have found that loss of the atrR gene eliminated the high-level azole resistance seen in clinical isolates. Additional, preliminary data generated by use of chromatin immunoprecipitation coupled with high throughput DNA sequencing (ChIP-seq) and RNA-seq have shed light on direct and indirect targets of AtrR regulation. We have generated two different hyperactive alleles of atrR that drive elevated expression of abcG1 and enhanced azole resistance. These data suggest that AtrR is normally subject to negative regulation that can be overcome in different manners. We suggest that defects in this negative regulatory system may influence clinically significant azole resistance owing to increased expression of AtrR-dependent target genes (like abcG1 and cyp51A). The goal of this proposal is to employ a combined biochemical, molecular biological and genetic dissection of the regulation of AtrR in order to understand how this factor acts to induce azole resistance. Our initial goal is to carry out a structure/function analysis of AtrR and identify protein domains that are important for regulation of this factor. We will also use direct biochemical purification to identify factors that associate with AtrR and influence its function. Second, we will use forward genetic screening involving impala transposon mutagenesis to identify AtrR regulatory factors in an unbiased, functional manner. Finally, we will examine the role of an AtrR target gene that encodes a transcription factor called RfeC. Preliminary data indicate that RfeC is important in AbcG1 expression and azole resistance. We will examine the epistatic relationship between our atrR alleles and rfeC (as well as other azole resistance-affecting transcription factors) to establish the regulatory hierarchy controlling azole resistance. These experiments will illuminate the physiological network controlling AtrR that directly links ergosterol biosynthesis to ABC transporter gene expression and provide important new information about azole resistance in this fungal pathogen.